Project Summary The objective of this proposal is to pioneer gene regulatory network studies of early Neural Crest (NC) specification at previously uncharacterized early stages of development. NC cells are unique to vertebrates, migrate extensively, and differentiate into multiple derivatives, including neurons, glia, melanocytes, odontoblasts, and most of the craniofacial skeleton and connective tissue. Their failed development and homeostasis leads to multiple pathologies (collectively termed ?neurocristopathies?) which include prevalent craniofacial malformations such as cleft lip and cleft palate. Traditionally, NC is thought to be induced from ectoderm-derived cells at the neural plate border, as a result of inductive interactions involving neural and non- neural ectoderm and/or underlying mesoderm. However, recent evidence from the Garcia-Castro lab and others using chick, Xenopus, and human embryonic stem cell models suggest an earlier, pre-gastrula origin of NC that is independent from post-gastrula tissue contributions. Yet the molecular mechanisms underlying this early NC-specification events remain uncharacterized, and this proposal precisely aims to address this issue. The evidence from the Garcia-Castro lab demonstrated the formation of human NC in a human pluripotent stem cell model, independent of mesodermal or neural contributions, and suggested an earlier than anticipated stage of specification, preceding the accepted neural plate border stage. Importantly, very similar conclusions where reached in the chick embryo through specification assays and fate mapping experiments. This suggests that the NC lineage rapidly departs from a pluripotent state to become specified and engaged (not committed) in the NC lineage. Our group specifically recognizes this as a novel stage we termed pre-border. Transcriptomic profiling of hNC supports an early stage, characterized by a set of transcription factors, whose expression is rapidly initiated, precedes that of commonly used NC markers known as neural plate border specifiers, further supporting our proposed pre-border stage. Here, I hypothesize that the transcription factors expressed in the pre-border stage function in the early activation of genes known to operate at later stages in the NC-gene regulatory network. Therefore, I propose to assess the contributions of a selected group of pre-border factors to the regulation of the NC lineage, by determining their effects on known NC factors (knockdown and knockout strategies), and monitoring global changes of expression to identify other regulated molecules (Aim1) Importantly, comparative efforts in chick embryos will further strengthen these studies. Then I propose to determine the direct contributions of the pre-border factors through the identification of the cis regulatory modules to which they bind to regulate the expression of modulated targets (Aim2). This proposal will reveal for the first time the regulatory interactions between TFs responsible for the pre-border stage and its contribution to the formation of the neural crest.